G protein coupled receptors (hereinafter sometimes termed "GPCR"s) comprise a large superfamily of receptors sharing a common structural motif of seven transmembrane helical domains. When a ligand (an agonist) binds to a GPCR and activates it, signal transduction is achieved through the intermediary G protein (a heterotrimeric GTP binding protein) which in turn activates the second messenger system. Although the exact nature of the receptor-G protein interactions is not yet known, the receptor activated regulatory cycle of the G protein involves GTP exchange for GDP, dissociation of the .alpha. and .beta..gamma. subunits, activation of the second messenger pathway by GTP-G.sub.60 and .beta..gamma., and termination of activation upon GTP hydrolysis to GDP by the inherent GPTase activity of the .alpha. subunit. G protein coupled receptors regulate virtually all bodily functions ranging from vision and olfaction to neuronal and endocrine signaling.
A general property of signal transduction mediated by G protein coupled receptors is the attenuation of signaling upon prolonged agonist stimulation. These processes are referred to as desensitization, tachyphylaxis, adaptation, tolerance, or quenching. Because signal attenuation limits the clinical uses of many pharmaceuticals acting on GPCRs, the mechanism for this process has been the focus of much research. Receptor phosphorylation by selective kinases of G protein coupled receptors (termed "GRK"s) has been shown to contribute to desensitization of several receptors. To date, no selective and or potent GRK inhibitors have been reported, other than heparin which does not penetrate into intact cells, even though such inhibitors might prevent desensitization in these cases (e.g., the .beta.2 receptor). GRKs selectively polyphosphorylate only the active receptor state, which not only serves as a preferred substrate, but also directly stimulates GRK activity.
Another emerging recognized feature of a number of GPCRs is the presence of a basal level of signaling activity, occurring in the absence of any agonist ligand. Mutations inducing high basal activity have been associated with genetic disorders, demonstrating the physiological relevance of basal receptor activity. For GPCRs displaying basal activity, two classes of antagonist have been defined, i.e., neutral antagonists which block only agonist induced effects without changing basal activity, and inverse agonists, or negative antagonists (or inverse agonists), which also block basal receptor activity.
Prior assay attempts to detect any significant changes of the .mu. opioid receptor system during prolonged agonist exposure have been unable to determine biochemical mechanisms underlying narcotic addiction. Thus, much of the current research work has focused on events downstream of the receptor, such as long-term gene regulation, in attempting to account for the dependent-tolerant state. Because tolerance and the dependence liability of narcotic drugs severely limit their clinical utility as potent analgesics and exert a heavy toll on society through illicit narcotic drug use, a screen for agents that could prevent or reverse the narcotic dependent-tolerant state or might facilitate gradual withdrawal would greatly enhance the clinical utility of narcotic analgesics and could serve as an effective pharmacological weapon in the fight against illicit drug use.